Microorganism concentration process and agent

ABSTRACT

A process for capturing or concentrating microorganisms for detection or assay comprises (a) providing a concentration agent that comprises diatomaceous earth bearing, on at least a portion of its surface, a surface treatment comprising a surface modifier comprising titanium dioxide, fine-nanoscale gold or platinum, or a combination thereof; (b) providing a sample comprising at least one microorganism strain; and (c) contacting the concentration agent with the sample such that at least a portion of the at least one microorganism strain is bound to or captured by the concentration agent.

STATEMENT OF PRIORITY

This application is a filing under 35 U.S.C. 371, of InternationalApplication No. PCT/US08/78575, filed Oct. 2, 2008; and which claims thepriority of U.S. Provisional Application No. 60/977,200 filed Oct. 3,2007, the contents of which are hereby incorporated by reference.

FIELD

This invention relates to processes for capturing or concentratingmicroorganisms such that they remain viable for detection or assay. Inother aspects, this invention also relates to concentration agents (andmethods for their preparation) and diagnostic kits for use in carryingout such concentration processes.

BACKGROUND

Food-borne illnesses and hospital-acquired infections resulting frommicroorganism contamination are a concern in numerous locations all overthe world. Thus, it is often desirable or necessary to assay for thepresence of bacteria or other microorganisms in various clinical, food,environmental, or other samples, in order to determine the identityand/or the quantity of the microorganisms present.

Bacterial DNA or bacterial RNA, for example, can be assayed to assessthe presence or absence of a particular bacterial species even in thepresence of other bacterial species. The ability to detect the presenceof a particular bacterium, however, depends, at least in part, on theconcentration of the bacterium in the sample being analyzed. Bacterialsamples can be plated or cultured to increase the numbers of thebacteria in the sample to ensure an adequate level for detection, butthe culturing step often requires substantial time and therefore cansignificantly delay the assessment results.

Concentration of the bacteria in the sample can shorten the culturingtime or even eliminate the need for a culturing step. Thus, methods havebeen developed to isolate (and thereby concentrate) particular bacterialstrains by using antibodies specific to the strain (for example, in theform of antibody-coated magnetic or non-magnetic particles). Suchmethods, however, have tended to be expensive and still somewhat slowerthan desired for at least some diagnostic applications.

Concentration methods that are not strain-specific have also been used(for example, to obtain a more general assessment of the microorganismspresent in a sample). After concentration of a mixed population ofmicroorganisms, the presence of particular strains can be determined, ifdesired, by using strain-specific probes.

Non-specific concentration or capture of microorganisms has beenachieved through methods based upon carbohydrate and lectin proteininteractions. Chitosan-coated supports have been used as non-specificcapture devices, and substances (for example, carbohydrates, vitamins,iron-chelating compounds, and siderophores) that serve as nutrients formicroorganisms have also been described as being useful as ligands toprovide non-specific capture of microorganisms.

Various inorganic materials (for example, hydroxyapatite and metalhydroxides) have been used to non-specifically bind and concentratebacteria. Physical concentration methods (for example, filtration,chromatography, centrifugation, and gravitational settling) have alsobeen utilized for non-specific capture, with and/or without the use ofinorganic binding agents. Such non-specific concentration methods havevaried in speed, cost (at least some requiring expensive equipment,materials, and/or trained technicians), sample requirements (forexample, sample nature and/or volume limitations), space requirements,ease of use (at least some requiring complicated multi-step processes),suitability for on-site use, and/or effectiveness.

SUMMARY

Thus, we recognize that there is an urgent need for processes forrapidly detecting pathogenic microorganisms. Such processes willpreferably be not only rapid but also low in cost, simple (involving nocomplex equipment or procedures), and/or effective under a variety ofconditions (for example, with varying types of sample matrices, varyingbacterial loads, and varying sample volumes).

Briefly, in one aspect, this invention provides a process fornon-specifically concentrating the strains of microorganisms (forexample, strains of bacteria, fungi, yeasts, protozoans, viruses(including both non-enveloped and enveloped viruses), and bacterialendospores) present in a sample, such that the microorganisms remainviable for the purpose of detection or assay of one or more of thestrains. The process comprises (a) providing a concentration agent(preferably, a particulate concentration agent) that comprisesdiatomaceous earth bearing, on at least a portion of its surface, asurface treatment comprising a surface modifier comprising titaniumdioxide, fine-nanoscale gold or platinum, or a combination thereof; (b)providing a sample (preferably, in the form of a fluid) comprising atleast one microorganism strain; and (c) contacting (preferably, bymixing) the concentration agent with the sample such that at least aportion of the at least one microorganism strain is bound to or capturedby the concentration agent. Preferably, the process further comprisesdetecting the presence of the at least one bound microorganism strain(for example, by culture-based, microscopy/imaging, genetic,bioluminescence-based, or immunologic detection methods) and/orsegregating (preferably, by gravitational settling) the resultingmicroorganism-bound concentration agent. The process can optionallyfurther comprise separating the resulting segregated concentration agentfrom the sample.

The process of the invention does not target a specific microorganismstrain. Rather, it has been discovered that certain relativelyinexpensive, inorganic materials can be surprisingly effective incapturing a variety of microorganisms. Such materials can be used toconcentrate the microorganism strains present in a sample (for example,a food sample) in a non-strain-specific manner, so that one or more ofthe microorganism strains (preferably, one or more strains of bacteria)can be more easily and rapidly assayed.

The process of the invention is relatively simple and low in cost(requiring no complex equipment or expensive strain-specific materials)and can be relatively fast (preferred embodiments capturing at leastabout 70 percent (more preferably, at least about 80 percent; mostpreferably, at least about 90 percent) of the microorganisms present ina sample in less than about 30 minutes, relative to a correspondingcontrol sample without concentration agent). In addition, the processcan be effective with a variety of microoganisms (including pathogenssuch as both gram positive and gram negative bacteria) and with avariety of samples (different sample matrices and, unlike at least someprior art methods, even samples having low microorganism content and/orlarge volumes). Thus, at least some embodiments of the process of theinvention can meet the above-cited urgent need for low-cost, simpleprocesses for rapidly detecting pathogenic microorganisms under avariety of conditions.

In another aspect, the invention also provides a diagnostic kit for usein carrying out the process of the invention, the kit comprising (a) aconcentration agent (preferably, a particulate concentration agent) thatcomprises diatomaceous earth bearing, on at least a portion of itssurface, a surface treatment comprising a surface modifier comprisingtitanium dioxide, fine-nanoscale gold or platinum, or a combinationthereof; and (b) a testing container (preferably, a sterile testingcontainer) for use in carrying out the process of the invention.Preferably, the diagnostic kit further comprises one or more componentsselected from microorganism culture media, lysis reagents, buffers,genetic detection assay components, bioluminescence detection assaycomponents, instructions for carrying out the process, and combinationsthereof.

In yet another aspect, the invention provides a concentration agentcomprising titanium dioxide, fine-nanoscale gold or platinum, or acombination thereof on a particulate support selected from diatomaceousearth, metal oxide-modified diatomaceous earth, and combinationsthereof.

In still another aspect, the invention provides a process for preparinga concentration agent comprising (a) providing a particulate supportselected from diatomaceous earth, metal oxide-modified diatomaceousearth, and combinations thereof; and (b) depositing fine-nanoscale goldor platinum on the support by physical vapor deposition.

In yet another aspect, the invention provides a process for preparing aconcentration agent comprising (a) providing a particulate supportselected from diatomaceous earth, metal oxide-modified diatomaceousearth, and combinations thereof; (b) providing a hydrolyzable titaniumdioxide precursor compound; (c) combining the support and the compound;and (d) hydrolyzing the compound so as to deposit titanium dioxide onthe support.

BRIEF DESCRIPTION OF DRAWING

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawing, wherein:

FIG. 1 shows, in side sectional view, an apparatus that was used inpreparing concentration agents for use in carrying out the embodimentsof the process of the invention described in the examples section below.

FIG. 2 shows, in perspective view, the apparatus of FIG. 1.

These figures, which are idealized, are not drawn to scale and areintended to be merely illustrative and nonlimiting.

DETAILED DESCRIPTION Definitions

As used in this patent application:

“sample” means a substance or material that is collected (for example,to be analyzed);

“sample matrix” means the components of a sample other thanmicroorganisms;

“detection” means the identification of at least a component of amicroorganism, which thereby determines that the microorganism ispresent;

“genetic detection” means the identification of a component of geneticmaterial such as DNA or RNA that is derived from a target microorganism;

“immunologic detection” means the identification of an antigenicmaterial such as a protein or a proteoglycan that is derived from atarget microorganism;

“microorganism” means any cell having genetic material suitable foranalysis or detection (including, for example, bacteria, yeasts,viruses, and bacterial endospores);

“microorganism strain” means a particular type of microorganism that isdistinguishable through a detection method (for example, microorganismsof different genera, of different species within a genera, or ofdifferent isolates within a species); and

“target microorganism” means any microorganism that is desired to bedetected.

Concentration Agent

Concentration agents suitable for use in carrying out the process of theinvention include those that comprise diatomaceous earth bearing, on atleast a portion of its surface, a surface treatment comprising a surfacemodifier comprising titanium dioxide, fine-nanoscale gold or platinum,or a combination thereof. Concentration or capture using suchconcentration agents is generally not specific to any particular strain,species, or type of microorganism and therefore provides for theconcentration of a general population of microorganisms in a sample.Specific strains of microorganisms can then be detected from among thecaptured microorganism population using any known detection method withstrain-specific probes. Thus, the concentration agents can be used forthe detection of microbial contaminants or pathogens (particularlyfood-borne pathogens such as bacteria) in clinical, food, environmental,or other samples.

In carrying out the process of the invention, the concentration agentscan be used in any form that is amenable to sample contact andmicroorganism capture (for example, in particulate form or applied to asupport such as a dipstick, film, filter, tube, well, plate, beads,membrane, or channel of a microfluidic device, or the like). Preferably,the concentration agents are used in particulate form.

When dispersed or suspended in water systems, inorganic materialsexhibit surface charges that are characteristic of the material and thepH of the water system. The potential across the material-waterinterface is called the “zeta potential,” which can be calculated fromelectrophoretic mobilities (that is, from the rates at which theparticles of material travel between charged electrodes placed in thewater system). At least some of the concentration agents used incarrying out the process of the invention have zeta potentials that areat least somewhat more positive than that of untreated diatomaceousearth, and the concentration agents can be surprisingly significantlymore effective than untreated diatomaceous earth in concentratingmicroorganisms such as bacteria, the surfaces of which generally tend tobe negatively charged. Preferably, the concentration agents have anegative zeta potential at a pH of about 7 (more preferably, a zetapotential in the range of about −5 millivolts to about −20 millivolts ata pH of about 7; even more preferably, a zeta potential in the range ofabout −8 millivolts to about −19 millivolts at a pH of about 7; mostpreferably, a zeta potential in the range of about −10 millivolts toabout −18 millivolts at a pH of about 7).

Thus, it has been discovered that concentration agents comprisingcertain types of surface-treated or surface-modified diatomaceous earth(namely, bearing a surface treatment comprising a surface modifiercomprising titanium dioxide, fine-nanoscale gold or platinum, or acombination thereof) can be surprisingly more effective than untreateddiatomaceous earth in concentrating microorganisms. The surfacetreatment preferably further comprises a metal oxide selected fromferric oxide, zinc oxide, aluminum oxide, and the like, and combinationsthereof (more preferably, ferric oxide). Although noble metals such asgold have been known to exhibit antimicrobial characteristics, thegold-containing concentration agents used in the process of theinvention surprisingly can be effective not only in concentrating themicroorganisms but also in leaving them viable for purposes of detectionor assay.

Useful surface modifiers include fine-nanoscale gold; fine-nanoscaleplatinum; fine-nanoscale gold in combination with at least one metaloxide (preferably, titanium dioxide, ferric oxide, or a combinationthereof); titanium dioxide; titanium dioxide in combination with atleast one other (that is, other than titanium dioxide) metal oxide; andthe like; and combinations thereof. Preferred surface modifiers includefine-nanoscale gold; fine-nanoscale platinum; fine-nanoscale gold incombination with at least ferric oxide or titanium dioxide; titaniumdioxide; titanium dioxide in combination with at least ferric oxide; andcombinations thereof.

More preferred surface modifiers include fine-nanoscale gold;fine-nanoscale platinum; fine-nanoscale gold in combination with ferricoxide or titanium dioxide; titanium dioxide; titanium dioxide incombination with ferric oxide; and combinations thereof (even morepreferably, fine-nanoscale gold; fine-nanoscale gold in combination withferric oxide or titanium dioxide; titanium dioxide in combination withferric oxide; and combinations thereof). Fine-nanoscale gold,fine-nanoscale gold in combination with ferric oxide or titaniumdioxide, and combinations thereof are most preferred.

Gold and/or Platinum

The concentration agents comprising fine-nanoscale gold or platinum canbe prepared by depositing gold or platinum on diatomaceous earth byphysical vapor deposition (optionally, by physical vapor deposition inan oxidizing atmosphere). As used herein, the term “fine-nanoscale goldor platinum” refers to gold or platinum bodies (for example, particlesor atom clusters) having all dimensions less than or equal to 5nanometers (nm) in size. Preferably, at least a portion of the depositedgold or platinum has all dimensions (for example, particle diameter oratom cluster diameter) in the range of up to (less than or equal to)about 10 nm in average size (more preferably, up to about 5 nm; evenmore preferably, up to about 3 nm).

In most preferred embodiments, at least a portion of the gold isultra-nanoscale (that is, having at least two dimensions less than 0.5nm in size and all dimensions less than 1.5 nm in size). The size ofindividual gold or platinum nanoparticles can be determined bytransmission electron microscopy (TEM) analysis, as is well known in theart.

The amount of gold or platinum provided on the diatomaceous earth canvary over a wide range. Since gold and platinum are expensive, it isdesirable not to use more than is reasonably needed to achieve a desireddegree of concentration activity. Additionally, because nanoscale goldor platinum can be highly mobile when deposited using PVD, activity canbe compromised if too much gold or platinum is used, due to coalescenceof at least some of the gold or platinum into large bodies.

For these reasons, the weight loading of gold or platinum on thediatomaceous earth preferably is in the range of about 0.005 (morepreferably, 0.05) to about 10 weight percent, more preferably about0.005 (even more preferably, 0.05) to about 5 weight percent, and evenmore preferably from about 0.005 (most preferably, 0.05) to about 2.5weight percent, based upon the total weight of the diatomaceous earthand the gold or platinum.

Gold and platinum can be deposited by PVD techniques (for example, bysputtering) to form concentration-active, fine-nanoscale particles oratom clusters on a support surface. It is believed that the metal isdeposited mainly in elemental form, although other oxidation states maybe present.

In addition to gold and/or platinum, one or more other metals can alsobe provided on the same diatomaceous earth supports and/or on othersupports intermixed with the gold- and/or platinum-containing supports.Examples of such other metals include silver, palladium, rhodium,ruthenium, osmium, copper, iridium, and the like, and combinationsthereof. If used, these other metals can be co-deposited on a supportfrom a target source that is the same or different from the gold orplatinum source target that is used. Alternatively, such metals can beprovided on a support either before or after the gold and/or platinum isdeposited. Metals requiring a thermal treatment for activationadvantageously can be applied to a support and heat treated before thegold and/or platinum is deposited.

Physical vapor deposition refers to the physical transfer of metal froma metal-containing source or target to a support medium. Physical vapordeposition can be viewed as involving atom-by-atom deposition, althoughin actual practice the metal can be transferred as extremely fine bodiesconstituting more than one atom per body. The deposited metal caninteract with the surface of the support medium physically, chemically,ionically, and/or otherwise.

Physical vapor deposition preferably occurs under temperature and vacuumconditions in which the metal is quite mobile and will tend to migrateon the surface of the support medium until immobilized in some fashion(for example, by adhering to a site on or very near the supportsurface). Sites of adhering can include defects such as surfacevacancies, structural discontinuities such as steps and dislocations,and interfacial boundaries between phases or crystals or other metalspecies such as small metal clusters. Metal deposited by PVD apparentlyis sufficiently immobilized that the metal can retain a high level ofactivity. In contrast, conventional methodologies often allow the metalto coalesce into such large bodies that activity can be compromised oreven lost.

Physical vapor deposition can be carried out in various different ways.Representative approaches include sputter deposition (preferred),evaporation, and cathodic arc deposition. Any of these or other PVDapproaches can be used in preparing the concentration agents used incarrying out the process of the invention, although the nature of thePVD technique can impact the resulting activity.

For example, the energy of the physical vapor deposition technique canimpact the mobility of the deposited metal and hence its tendency tocoalesce. Higher energy tends to correspond to an increased tendency ofthe metal to coalesce. Increased coalescence, in turn, tends to reduceactivity. Generally, the energy of the depositing species is lowest forevaporation, higher for sputter deposition (which can include some ioncontent in which a small fraction of the impinging metal species areionized), and highest for cathodic arc deposition (which can includeseveral tens of percents of ion content). Accordingly, if a particularPVD technique yields deposited metal that is more mobile than desired,it can be useful to use a PVD technique of lesser energy instead.

Physical vapor deposition preferably is performed while the supportmedium to be treated is being well-mixed (for example, tumbled,fluidized, milled, or the like) to ensure adequate treatment of supportsurfaces. Methods of tumbling particles for deposition by PVD aredescribed in U.S. Pat. No. 4,618,525 (Chamberlain et al.), thedescriptions of which are incorporated herein by reference.

When carrying out PVD on fine particles or fine particle agglomerates(for example, less than about 10 micrometers in average diameter), thesupport medium is preferably both mixed and comminuted (for example,ground or milled to some degree) during at least a portion of the PVDprocess. This can assist in maintaining the separation and free flow ofthe particles or agglomerates during the deposition. In the case of fineparticles or fine particle agglomerates, it can be advantageous for themixing of the particles to be as vigorous and rapid as possible whilestill retaining controlled deposition of the metal.

PVD can be carried out by using any of the types of apparatus that arenow used or hereafter developed for this purpose. A preferred apparatus10 is shown, however, in FIGS. 1 and 2. The apparatus 10 includes ahousing 12 defining a vacuum chamber 14 containing a particle agitator16. The housing 12, which can be made from an aluminum alloy if desired,is a vertically oriented hollow cylinder (for example, 45 cm high and 50cm in diameter). The base 18 contains a port 20 for a high vacuum gatevalve 22 followed by a six-inch diffusion pump 24 as well as a support26 for the particle agitator 16. The vacuum chamber 14 is capable ofbeing evacuated to background pressures in the range of 10⁻⁶ Torr.

The top of the housing 12 includes a demountable, rubber L-gasket-sealedplate 28 that is fitted with an external mount, three-inch diameterdirect current (dc) magnetron sputter deposition source 30 (a US Gun II,US, INC., San Jose, Calif.). Into the sputter deposition source 30 isfastened a gold or platinum sputter target 32 (for example, 7.6 cm (3.0inch) diameter×0.48 cm ( 3/16 inch) thick). The sputter depositionsource 30 is powered by an MDX-10 Magnetron Drive (Advanced EnergyIndustries, Inc, Fort Collins, Colo.) fitted with a Sparc-le 20 arcsuppression system (Advanced Energy Industries, Inc, Fort Collins,Colo.).

The particle agitator 16 is a hollow cylinder (for example, 12 cmlong×9.5 cm diameter horizontal) with a rectangular opening 34 (forexample, 6.5 cm×7.5 cm). The opening 34 is positioned about 7 cmdirectly below the surface 36 of the sputter target 32, so thatsputtered metal atoms can enter the agitator volume 38. The agitator 16is fitted with a shaft 40 aligned with its axis. The shaft 40 has arectangular cross section (for example, 1 cm×1 cm) to which are boltedfour rectangular blades 42 which form an agitation mechanism or paddlewheel for the support particles being tumbled. The blades 42 eachcontain two holes 44 (for example, 2 cm diameter) to promotecommunication between the particle volumes contained in each of the fourquadrants formed by the blades 42 and particle agitator 16. Thedimensions of the blades 42 are selected to give side and end gapdistances of either 2.7 mm or 1.7 mm with the agitator walls 48.

Physical vapor deposition can be carried out at essentially any desiredtemperature(s) over a very wide range. However, the deposited metal canbe more active (perhaps due to more defects and/or lower mobility andcoalescence) if the metal is deposited at relatively low temperatures(for example, at a temperature below about 150° C., preferably belowabout 50° C., more preferably at ambient temperature (for example, about20° C. to about 27° C.) or less). Operating under ambient conditions canbe generally preferred as being effective and economical, as no heatingor chilling is required during the deposition.

The physical vapor deposition can be carried out in an inert sputteringgas atmosphere (for example, in argon, helium, xenon, radon, or amixture of two or more thereof (preferably, argon)), and, optionally,the physical vapor deposition can be carried out in an oxidizingatmosphere. The oxidizing atmosphere preferably comprises at least oneoxygen-containing gas (more preferably, an oxygen-containing gasselected from oxygen, water, hydrogen peroxide, ozone, and combinationsthereof; even more preferably, an oxygen-containing gas selected fromoxygen, water, and combinations thereof; most preferably, oxygen). Theoxidizing atmosphere further comprises an inert sputtering gas such asargon, helium, xenon, radon, or a mixture of two or more thereof(preferably, argon). The total gas pressure (all gases) in the vacuumchamber during the PVD process can be from about 1 mTorr to about 25mTorr (preferably, from about 5 mTorr to about 15 mTorr). The oxidizingatmosphere can comprise from about 0.05 percent to about 60 percent byweight oxygen-containing gas (preferably, from about 0.1 percent toabout 50 percent by weight; more preferably, from about 0.5 percent toabout 25 percent by weight), based upon the total weight of all gases inthe vacuum chamber.

The diatomaceous earth support medium can optionally be calcined priorto metal deposition, although this can increase its crystalline silicacontent. Since gold and platinum are active right away when depositedvia PVD, there is generally no need for heat treatment after metaldeposition, unlike deposition by some other methodologies. Such heattreating or calcining can be carried out if desired, however, to enhanceactivity.

In general, thermal treatment can involve heating the support at atemperature in the range of about 125° C. to about 1000° C. for a timeperiod in the range of about 1 second to about 40 hours, preferablyabout 1 minute to about 6 hours, in any suitable atmosphere such as air,an inert atmosphere such as nitrogen, carbon dioxide, argon, a reducingatmosphere such as hydrogen, and the like. The particular thermalconditions to be used can depend upon various factors including thenature of the support.

Generally, thermal treatment can be carried out below a temperature atwhich the constituents of the support would be decomposed, degraded, orotherwise unduly thermally damaged. Depending upon factors such as thenature of the support, the amount of metal, and the like, activity canbe compromised to some degree if the system is thermally treated at toohigh a temperature.

Titanium Dioxide and/or Other Metal Oxides

The concentration agents comprising metal oxide can be prepared bydepositing metal oxide on diatomaceous earth by hydrolysis of ahydrolyzable metal oxide precursor compound. Suitable metal oxideprecursor compounds include metal complexes and metal salts that can behydrolyzed to form metal oxides. Useful metal complexes include thosecomprising alkoxide ligands, hydrogen peroxide as a ligand,carboxylate-functional ligands, and the like, and combinations thereof,Useful metal salts include metal sulfates, nitrates, halides,carbonates, oxalates, hydroxides, and the like, and combinationsthereof.

When using metal salts or metal complexes of hydrogen peroxide orcarboxylate-functional ligands, hydrolysis can be induced by eitherchemical or thermal means. In chemically-induced hydrolysis, the metalsalt can be introduced in the form of a solution into a dispersion ofthe diatomaceous earth, and the pH of the resulting combination can beraised by the addition of a base solution until the metal saltprecipitates as a hydroxide complex of the metal on the diatomaceousearth. Suitable bases include alkali metal and alkaline earth metalhydroxides and carbonates, ammonium and alkyl-ammonium hydroxides andcarbonates, and the like, and combinations thereof. The metal saltsolution and the base solution can generally be about 0.1 to about 2 Min concentration.

Preferably, the addition of the metal salt to the diatomaceous earth iscarried out with stirring (preferably, rapid stirring) of thediatomaceous earth dispersion. The metal salt solution and the basesolution can be introduced to the diatomaceous earth dispersionseparately (in either order) or simultaneously, so as to effect apreferably substantially uniform reaction of the resulting metalhydroxide complex with the surface of the diatomaceous earth. Thereaction mixture can optionally be heated during the reaction toaccelerate the speed of the reaction. In general, the amount of baseadded can equal the number of moles of the metal times the number ofnon-oxo and non-hydroxo counterions on the metal salt or metal complex.

Alternatively, when using salts of titanium or iron, the metal salt canbe thermally induced to hydrolyze to form the hydroxide complex of themetal and to interact with the surface of the diatomaceous earth. Inthis case, the metal salt solution can generally be added to adispersion of the diatomaceous earth (preferably, a stirred dispersion)that has been heated to a sufficiently high temperature (for example,greater than about 50° C.) to promote the hydrolysis of the metal salt.Preferably, the temperature is between about 75° C. and 100° C.,although higher temperatures can be used if the reaction is carried outin an autoclave apparatus.

When using metal alkoxide complexes, the metal complex can be induced tohydrolyze to form a hydroxide complex of the metal by partial hydrolysisof the metal alkoxide in an alcohol solution. Hydrolysis of the metalalkoxide solution in the presence of diatomaceous earth can result inmetal hydroxide species being deposited on the surface of thediatomaceous earth.

Alternatively, the metal alkoxide can be hydrolyzed and deposited ontothe surface of the diatomaceous earth by reacting the metal alkoxide inthe gas phase with water, in the presence of the diatomaceous earth. Inthis case, the diatomaceous earth can be agitated during the depositionin either, for example, a fluidized bed reactor or a rotating drumreactor.

After the above-described hydrolysis of the metal oxide precursorcompound in the presence of the diatomaceous earth, the resultingsurface-treated diatomaceous earth can be separated by settling or byfiltration or by other known techniques. The separated product can bepurified by washing with water and can then be dried (for example, at50° C. to 150° C.).

Although the surface-treated diatomaceous earth generally can befunctional after drying, it can optionally be calcined to removevolatile by-products by heating in air to about 250° C. to 650° C.generally without loss of function. This calcining step can be preferredwhen metal alkoxides are utilized as the metal oxide precursorcompounds.

In general, with metal oxide precursor compounds of iron, the resultingsurface treatments comprise nanoparticulate iron oxide. When the weightratio of iron oxide to diatomaceous earth is about 0.08, X-raydiffraction (XRD) does not show the presence of a well-defined ironoxide material. Rather, additional X-ray reflections are observed at3.80, 3.68, and 2.94 Å. TEM examination of this material shows thesurface of the diatomaceous earth to be relatively uniformly coated withglobular nanoparticulate iron oxide material. The crystallite size ofthe iron oxide material is less than about 20 nm, with most of thecrystals being less than about 10 nm in diameter. The packing of theseglobular crystals on the surface of the diatomaceous earth is dense inappearance, and the surface of the diatomaceous earth appears to beroughened by the presence of these crystals.

In general, with metal oxide precursor compounds of titanium, theresulting surface treatments comprise nanoparticulate titania. Whendepositing titanium dioxide onto diatomaceous earth, XRD of theresulting product after calcination to about 350° C. can show thepresence of small crystals of anatase titania. With relatively lowertitanium/diatomaceous earth ratios or in cases where mixtures oftitanium and iron oxide precursors are used, no evidence of anatase isgenerally observed by X-ray analysis.

Since titania is well-known as a potent photo-oxidation catalyst, thetitania-modified diatomaceous earth concentration agents of the presentinvention can be used to concentrate microorganisms for analysis andthen optionally also be used as photoactivatable agents for killingresidual microorganisms and removing unwanted organic impurities afteruse. Thus, the titania-modified diatomaceous earth can both isolatebiomaterials for analysis and then be photochemically cleaned forre-use. These materials can also be used in filtration applicationswhere microorganism removal as well as antimicrobial effects can bedesired.

Diatomaceous Earth

Diatomaceous earth (or kieselguhr) is a natural siliceous materialproduced from the remnants of diatoms, a class of ocean-dwellingmicroorganisms. Thus, it can be obtained from natural sources and isalso commercially available (for example, from Alfa Aesar, A JohnsonMatthey Company, Ward Hill, Mass.). Diatomaceous earth particlesgenerally comprise small, open networks of silica in the form ofsymmetrical cubes, cylinders, spheres, plates, rectangular boxes, andthe like. The pore structures in these particles can generally beremarkably uniform.

Diatomaceous earth can be used in carrying out the process of theinvention as the raw, mined material or as purified and optionallymilled particles. Preferably, the diatomaceous earth is in the form ofmilled particles with sizes in the range of about 1 micrometer to about50 micrometers in diameter (more preferably, about 3 micrometers toabout 10 micrometers).

The diatomaceous earth can optionally be heat treated prior to use toremove any vestiges of organic residues. If a heat treatment is used, itcan be preferable that the heat treatment be at 500° C. or lower, ashigher temperatures can produce undesirably high levels of crystallinesilica.

Sample

The process of the invention can be applied to a variety of differenttypes of samples, including, but not limited to, medical, environmental,food, feed, clinical, and laboratory samples, and combinations thereof.Medical or veterinary samples can include, for example, cells, tissues,or fluids from a biological source (for example, a human or an animal)that are to be assayed for clinical diagnosis. Environmental samples canbe, for example, from a medical or veterinary facility, an industrialfacility, soil, a water source, a food preparation area (food contactand non-contact areas), a laboratory, or an area that has beenpotentially subjected to bioterrorism. Food processing, handling, andpreparation area samples are preferred, as these are often of particularconcern in regard to food supply contamination by bacterial pathogens.

Samples obtained in the form of a liquid or in the form of a dispersionor suspension of solid in liquid can be used directly, or can beconcentrated (for example, by centrifugation) or diluted (for example,by the addition of a buffer (pH-controlled) solution). Samples in theform of a solid or a semi-solid can be used directly or can beextracted, if desired, by a method such as, for example, washing orrinsing with, or suspending or dispersing in, a fluid medium (forexample, a buffer solution). Samples can be taken from surfaces (forexample, by swabbing or rinsing). Preferably, the sample is a fluid (forexample, a liquid, a gas, or a dispersion or suspension of solid orliquid in liquid or gas).

Examples of samples that can be used in carrying out the process of theinvention include foods (for example, fresh produce or ready-to-eatlunch or “deli” meats), beverages (for example, juices or carbonatedbeverages), potable water, and biological fluids (for example, wholeblood or a component thereof such as plasma, a platelet-enriched bloodfraction, a platelet concentrate, or packed red blood cells; cellpreparations (for example, dispersed tissue, bone marrow aspirates, orvertebral body bone marrow); cell suspensions; urine, saliva, and otherbody fluids; bone marrow; lung fluid; cerebral fluid; wound exudate;wound biopsy samples; ocular fluid; spinal fluid; and the like), as wellas lysed preparations, such as cell lysates, which can be formed usingknown procedures such as the use of lysing buffers, and the like.Preferred samples include foods, beverages, potable water, biologicalfluids, and combinations thereof (with foods, beverages, potable water,and combinations thereof being more preferred).

Sample volume can vary, depending upon the particular application. Forexample, when the process of the invention is used for a diagnostic orresearch application, the volume of the sample can typically be in themicroliter range (for example, 10 μL or greater). When the process isused for a food pathogen testing assay or for potable water safetytesting, the volume of the sample can typically be in the milliliter toliter range (for example, 100 milliliters to 3 liters). In an industrialapplication, such as bioprocessing or pharmaceutical formulation, thevolume can be tens of thousands of liters.

The process of the invention can isolate microorganisms from a sample ina concentrated state and can also allow the isolation of microorganismsfrom sample matrix components that can inhibit detection procedures thatare to be used. In all of these cases, the process of the invention canbe used in addition to, or in replacement of, other methods ofmicroorganism concentration. Thus, optionally, cultures can be grownfrom samples either before or after carrying out the process of theinvention, if additional concentration is desired.

Contacting

The process of the invention can be carried out by any of various knownor hereafter-developed methods of providing contact between twomaterials. For example, the concentration agent can be added to thesample, or the sample can be added to the concentration agent. Adipstick coated with concentration agent can be immersed in a samplesolution, a sample solution can be poured onto a film coated withconcentration agent, a sample solution can be poured into a tube or wellcoated with concentration agent, or a sample solution can be passedthrough a filter (for example, a woven or nonwoven filter) coated withconcentration agent.

Preferably, however, the concentration agent and the sample are combined(using any order of addition) in any of a variety of containers(optionally but preferably, a capped, closed, or sealed container; morepreferably, a capped test tube, bottle, or jar). Suitable containers foruse in carrying out the process of the invention will be determined bythe particular sample and can vary widely in size and nature. Forexample, the container can be small, such as a 10 microliter container(for example, a test tube) or larger, such as a 100 milliliter to 3liter container (for example, an Erlenmeyer flask or a polypropylenelarge-mouth bottle). The container, the concentration agent, and anyother apparatus or additives that contact the sample directly can besterilized (for example, by controlled heat, ethylene oxide gas, orradiation) prior to use, in order to reduce or prevent any contaminationof the sample that might cause detection errors. The amount ofconcentration agent that is sufficient to capture or concentrate themicroorganisms of a particular sample for successful detection will vary(depending upon, for example, the nature and form of the concentrationagent and sample volume) and can be readily determined by one skilled inthe art. For example, 10 milligrams of concentration agent permilliliter of sample can be useful for some applications.

If desired, contacting can be effected by passing a particulateconcentration agent at least once through a sample (for example, byrelying upon gravitational settling over a period of, for example, about10 minutes). Contact can be enhanced by mixing (for example, bystirring, shaking, or use of a rocking platform) such that the particlesof concentration agent repeatedly pass or settle through a substantialportion of the sample. For small volumes on the order of microliters(typically less than 0.5 milliliter), mixing can be rapid such as byvortexing or “nutation,” for example as described in U.S. Pat. No.5,238,812 (Coulter et al.), the description of which is incorporatedherein by reference. For larger volumes on the order of greater than orequal to 0.5 milliliters (typically 0.5 milliliter to 3 liters), mixingcan be achieved by gently tumbling the particulate concentration agentand the sample in an “end over end” fashion, for example as described inU.S. Pat. No. 5,576,185 (Coulter et al.), the description of which isincorporated herein by reference. Such tumbling can be accomplished, forexample, by means of a device configured to hold a test tube or othertype of reaction vessel and to slowly rotate the test tube or vessel inan “end over end” manner. Contacting can be carried out for a desiredperiod (for example, for sample volumes of about 100 milliliters orless, up to about 60 minutes of contacting can be useful; preferably,about 15 seconds to about 10 minutes or longer; more preferably, about15 seconds to about 5 minutes).

Thus, in carrying out the process of the invention, mixing (for example,agitation, rocking, or stirring) and/or incubation (for example, atambient temperature) are optional but preferred, in order to increasemicroorganism contact with the concentration agent. A preferredcontacting method includes both mixing (for example, for about 15seconds to about 5 minutes) and incubating (for example, for about 3minutes to about 30 minutes) a microorganism-containing sample(preferably, a fluid) with particulate concentration agent. If desired,one or more additives (for example, lysis reagents, bioluminescenceassay reagents, nucleic acid capture reagents (for example, magneticbeads), microbial growth media, buffers (for example, to moisten a solidsample), microbial staining reagents, washing buffers (for example, towash away unbound material), elution agents (for example, serumalbumin), surfactants (for example, Triton™ X-100 nonionic surfactantavailable from Union Carbide Chemicals and Plastics, Houston, Tex.),mechanical abrasion/elution agents (for example, glass beads), and thelike) can be included in the combination of concentration agent andsample.

If desired, the concentration agent (alone or in combination with, forexample, antimicrobial materials and/or with carrier materials in theform of liquids (for example, water or oils), solids (for example,fabrics, polymers, papers, or inorganic solids), gels, creams, foams, orpastes) can be applied to or rubbed against a non-porous or porous,solid, microorganism-contaminated or microorganism-contaminatablematerial or surface (for example, for use as a “cleaning” agent).Binders, stabilizers, surfactants, or other property modifiers can beutilized, if desired.

For such use, the concentration agent can be applied to woven ornonwoven fabrics and can be applied to disposable surfaces such aspaper, tissues, cotton swabs, as well as to a variety of absorbent andnonabsorbent materials. For example, the concentration agent can beincorporated into cloth or paper carrier materials for use as “cleaning”wipes. The concentration agent can be applied (for example, in the formof wipes or pastes comprising a carrier material) to solid surfaces, forexample, in home, day-care, industrial, and hospital settings, forcleansing toys, equipment, medical devices, work surfaces, and the like.When used for cleansing or other purposes, the sample can besimultaneously collected and contacted with the concentration agent in asingle step, if desired.

Segregation and/or Separation

Optionally but preferably, the process of the invention furthercomprises segregation of the resulting microorganism-bound concentrationagent. Such segregation preferably can be achieved by relying, at leastin part, upon gravitational settling (gravity sedimentation; forexample, over a time period of about 5 minutes to about 30 minutes). Insome cases, however, it can be desirable to accelerate segregation (forexample, by centrifugation or filtration) or to use combinations of anyof the segregation methods.

The process of the invention can optionally further comprise separatingthe resulting microorganism-bound concentration agent and the sample.For fluid samples, this can involve removal or separation of thesupernatant that results upon segregation. Separation of the supernatantcan be carried out by numerous methods that are well-known in the art(for example, by decanting or siphoning, so as to leave themicroorganism-bound concentration agent at the bottom of the containeror vessel utilized in carrying out the process).

The process of the invention can be carried out manually (for example,in a batch-wise manner) or can be automated (for example, to enablecontinuous or semi-continuous processing).

Detection

A variety of microorganisms can be concentrated and, optionally butpreferably, detected by using the process of the invention, including,for example, bacteria, fungi, yeasts, protozoans, viruses (includingboth non-enveloped and enveloped viruses), bacterial endospores (forexample, Bacillus (including Bacillus anthracis, Bacillus cereus, andBacillus subtilis) and Clostridium (including Clostridium botulinum,Clostridium difficile, and Clostridium perfringens)), and the like, andcombinations thereof (preferably, bacteria, yeasts, viruses, bacterialendospores, fungi, and combinations thereof; more preferably, bacteria,yeasts, viruses, bacterial endospores, and combinations thereof; evenmore preferably, bacteria, viruses, bacterial endospores, andcombinations thereof; most preferably, gram-negative bacteria,gram-positive bacteria, non-enveloped viruses (for example, norovirus,poliovirus, hepatitis A virus, rhinovirus, and combinations thereof),bacterial endospores, and combinations thereof). The process has utilityin the detection of pathogens, which can be important for food safety orfor medical, environmental, or anti-terrorism reasons. The process canbe particularly useful in the detection of pathogenic bacteria (forexample, both gram negative and gram positive bacteria), as well asvarious yeasts, molds, and mycoplasmas (and combinations of any ofthese).

Genera of target microorganisms to be detected include, but are notlimited to, Listeria, Escherichia, Salmonella, Campylobacter,Clostridium, Helicobacter, Mycobacterium, Staphylococcus, Shigella,Enterococcus, Bacillus, Neisseria, Shigella, Streptococcus, Vibrio,Yersinia, Bordetella, Borrelia, Pseudomonas, Saccharomyces, Candida, andthe like, and combinations thereof. Samples can contain a plurality ofmicroorganism strains, and any one strain can be detected independentlyof any other strain. Specific microorganism strains that can be targetsfor detection include Escherichia coli, Yersinia enterocolitica,Yersinia pseudotuberculosis, Vibrio cholerae, Vibrio parahaemolyticus,Vibrio vulnificus, Listeria monocytogenes, Staphylococcus aureus,Salmonella enterica, Saccharomyces cerevisiae, Candida albicans,Staphylococcal enterotoxin ssp, Bacillus cereus, Bacillus anthracis,Bacillus atrophaeus, Bacillus subtilis, Clostridium perfringens,Clostridium botulinum, Clostridium difficile, Enterobacter sakazakii,Pseudomonas aeruginosa, and the like, and combinations thereof(preferably, Staphylococcus aureus, Salmonella enterica, Saccharomycescerevisiae, Bacillus atrophaeus, Bacillus subtilis, Escherichia coli,human-infecting non-enveloped enteric viruses for which Escherichia colibacteriophage is a surrogate, and combinations thereof).

Microorganisms that have been captured or bound (for example, byadsorption) by the concentration agent can be detected by essentiallyany desired method that is currently known or hereafter developed. Suchmethods include, for example, culture-based methods (which can bepreferred when time permits), microscopy (for example, using atransmitted light microscope or an epifluorescence microscope, which canbe used for visualizing microorganisms tagged with fluorescent dyes) andother imaging methods, immunological detection methods, and geneticdetection methods. The detection process following microorganism captureoptionally can include washing to remove sample matrix components.

Immunological detection is detection of an antigenic material derivedfrom a target organism, which is commonly a biological molecule (forexample, a protein or proteoglycan) acting as a marker on the surface ofbacteria or viral particles. Detection of the antigenic materialtypically can be by an antibody, a polypeptide selected from a processsuch as phage display, or an aptamer from a screening process.

Immunological detection methods are well-known and include, for example,immunoprecipitation and enzyme-linked immunosorbent assay (ELISA).Antibody binding can be detected in a variety of ways (for example, bylabeling either a primary or a secondary antibody with a fluorescentdye, with a quantum dot, or with an enzyme that can producechemiluminescence or a colored substrate, and using either a platereader or a lateral flow device).

Detection can also be carried out by genetic assay (for example, bynucleic acid hybridization or primer directed amplification), which isoften a preferred method. The captured or bound microorganisms can belysed to render their genetic material available for assay. Lysismethods are well-known and include, for example, treatments such assonication, osmotic shock, high temperature treatment (for example, fromabout 50° C. to about 100° C.), and incubation with an enzyme such aslysozyme, glucolase, zymolose, lyticase, proteinase K, proteinase E, andviral enolysins.

Many commonly-used genetic detection assays detect the nucleic acids ofa specific microorganism, including the DNA and/or RNA. The stringencyof conditions used in a genetic detection method correlates with thelevel of variation in nucleic acid sequence that is detected. Highlystringent conditions of salt concentration and temperature can limit thedetection to the exact nucleic acid sequence of the target. Thusmicroorganism strains with small variations in a target nucleic acidsequence can be distinguished using a highly stringent genetic assay.Genetic detection can be based on nucleic acid hybridization where asingle-stranded nucleic acid probe is hybridized to the denaturednucleic acids of the microorganism such that a double-stranded nucleicacid is produced, including the probe strand. One skilled in the artwill be familiar with probe labels, such as radioactive, fluorescent,and chemiluminescent labels, for detecting the hybrid following gelelectrophoresis, capillary electrophoresis, or other separation method.

Particularly useful genetic detection methods are based on primerdirected nucleic acid amplification. Primer directed nucleic acidamplification methods include, for example, thermal cycling methods (forexample, polymerase chain reaction (PCR), reverse transcriptasepolymerase chain reaction (RT-PCR), and ligase chain reaction (LCR)), aswell as isothermal methods and strand displacement amplification (SDA)(and combinations thereof; preferably, PCR or RT-PCR). Methods fordetection of the amplified product are not limited and include, forexample, gel electrophoresis separation and ethidium bromide staining,as well as detection of an incorporated fluorescent label or radio labelin the product. Methods that do not require a separation step prior todetection of the amplified product can also be used (for example,real-time PCR or homogeneous detection).

Bioluminescence detection methods are well-known and include, forexample, adensosine triphosphate (ATP) detection methods including thosedescribed in U.S. Pat. No. 7,422,868 (Fan et al.), the descriptions ofwhich are incorporated herein by reference.

Since the process of the invention is non-strain specific, it provides ageneral capture system that allows for multiple microorganism strains tobe targeted for assay in the same sample. For example, in assaying forcontamination of food samples, it can be desired to test for Listeriamonocytogenes, Escherichia coli, and Salmonella all in the same sample.A single capture step can then be followed by, for example, PCR orRT-PCR assays using specific primers to amplify different nucleic acidsequences from each of these microorganism strains. Thus, the need forseparate sample handling and preparation procedures for each strain canbe avoided.

Diagnostic Kit

A diagnostic kit for use in carrying out the process of the inventioncomprises (a) an above-described concentration agent (preferably,particulate); and (b) a testing container (preferably, a sterile testingcontainer) for use in carrying out the process of the invention.Preferably, the diagnostic kit further comprises one or more componentsselected from microorganism culture or growth media, lysis reagents,buffers, bioluminescence detection assay components (for example,luminometer, lysis reagents, luciferase enzyme, enzyme substrate,reaction buffers, and the like), genetic detection assay components,instructions for carrying out the process, and combinations thereof. Apreferred lysis reagent is a lytic enzyme supplied in a buffer, andpreferred genetic detection assay components include one or more primersspecific for a target microorganism.

For example, a preferred embodiment of the diagnostic kit of theinvention contains a particulate concentration agent (for example, in asterile disposable container such as a glass or polypropylene vial), incombination with instructions for using said agent in carrying out theprocess of the invention (for example, by mixing the concentration agentwith a fluid sample to be analyzed, allowing the concentration agent tosettle by gravity, removing the resulting supernatant, and detecting thepresence of at least one concentration agent-bound target microorganismstrain). The concentration agent optionally can be hydrated in a smallvolume of buffer with preservative to improve stability during storageand transportation and/or can be contained/aliquotted in a tear-open,sealed pouch to prevent contamination. The concentration agent can be inthe form of a dispersion or suspension in a liquid or can be in powderform. Preferably, the diagnostic kit comprises pre-measured aliquots(for example, based upon sample volume) of particulate concentrationagent (more preferably, contained in one or more tear-open, sealedpouches).

EXAMPLES

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

Materials

Kieselguhr (diatomaceous earth) was purchased from Alfa Aesar (A JohnsonMatthey Company, Ward Hill, Mass.) as a white powder (325 mesh; allparticles less than 44 micrometers in size). This material was shown byX-ray diffraction (XRD) to contain amorphous silica along withcrystalline α-cristobalite and quartz. Calcined diatomaceous earth waspurchased from Solvadis, GmbH, Frankfurt, Germany (and observed tocomprise small rods about 5 to about 80 micrometers in length and about3 to about 8 micrometers in width, along with porous disks and diskfragments up to about 60 micrometers in primary length and asymmetricalfragments about 3 to about 60 micrometers in primary length). Thismaterial was shown by XRD to comprise predominantly α-cristobalite.

All microorganism cultures were purchased from The American Type CultureCollection (ATCC; Manassas, Va.).

Concentration agents comprising various different surface modifiers(namely, titanium dioxide; titanium dioxide in combination with ferricoxide; platinum; gold; and gold in combination with ferric oxide) wereprepared by surface treating the diatomaceous earth in the mannerdescribed below:

Gold Deposition

About 57-60 g of dried diatomaceous earth or metal oxide-modifieddiatomaceous earth support media (about 300 mL volume of powder) wasfurther dried in an oven at 150° C. for 24 hours to remove residualwater. The resulting dried sample was placed while hot into the PVDapparatus described above in the detailed description with the PVDapparatus having a particle agitator with a blade gap of 2.7 mm. Thevacuum chamber of the apparatus was then evacuated to a backgroundpressure of about 5×10⁻⁵ Torr, and gas comprising argon sputtering gaswas admitted to the chamber at a pressure of about 10 mTorr.

The metal deposition process was then carried out by applying power tothe cathode of the apparatus for a pre-set period of time, with itsparticle agitator shaft and holed blades being rotated at 4 rpm duringDC magnetron sputter coating of metal at a controlled power of 0.02 kW.The duration of sputter coating was 5 hours. After the sputter coatingwas completed, the vacuum chamber was vented with air to ambientconditions, and the resulting metal-coated sample was removed from thePVD apparatus and sieved through a 25 mesh (0.707 mm) screen to separatefine particulates generated during the process. The amount of metal thathad been deposited on the sample was determined by weighing (both beforeand after the deposition process) the metal sputtering target that wasutilized. In general, about 18 percent of the weight loss of the targetrepresented metal deposited on the sample (based on inductively coupledplasma analysis). From this information, the resulting amount of gold onthe support medium was calculated to be about 0.9 weight percent.

Platinum Deposition

The above-described gold deposition process was essentially repeated,with the exception that a 7.62-cm (3-inch) platinum target wassubstituted for the 7.62-cm (3-inch) gold target that had been used, thepower was set at 0.03 kW, and the time of deposition was 1 hour. Theresulting amount of platinum on the support medium was calculated to beabout 0.25 weight percent.

Deposition of Titanium Dioxide

A 20 weight percent titanium (IV) oxysulfate dehydrate solution wasprepared by dissolving 20.0 g of TiO(SO₄).2H₂O (Noah TechnologiesCorporation, San Antonio, Tex.) in 80.0 g of deionized water withstirring. 50.0 g of this solution was mixed with 175 mL of deionizedwater to form a titanium dioxide precursor compound solution. Adispersion of diatomaceous earth was prepared by dispersing 50.0 g ofdiatomaceous earth in 500 mL of deionized water in a large beaker withrapid stirring. After heating the diatomaceous earth dispersion to about80° C., the titanium dioxide precursor compound solution was addeddropwise while rapidly stirring over a period of about 1 hour. After theaddition, the beaker was covered with a watch glass and its contentsheated to boiling for 20 minutes. An ammonium hydroxide solution wasadded to the beaker until the pH of the contents was about 9. Theresulting product was washed by settling/decantation until the pH of thewash water was neutral. The product was separated by filtration anddried overnight at 100° C.

A portion of the dried product was placed into a porcelain crucible andcalcined by heating from room temperature to 350° C. at a heating rateof about 3° C. per minute and then held at 350° C. for 1 hour.

Deposition of Iron Oxide

Iron oxide was deposited onto diatomaceous earth using essentially theabove-described titanium dioxide deposition process, with the exceptionthat a solution of 20.0 g of Fe(NO₃)₃.9H₂O (J. T. Baker, Inc.,Phillipsburg, N.J.) dissolved in 175 mL of deionized water wassubstituted for the titanyl sulfate solution. A portion of the resultingiron oxide-modified diatomaceous earth was similarly calcined to 350° C.for further testing.

Deposition of Iron Oxide and Titanium Dioxide

A mixture of iron oxide and titanium dioxide was deposited ontodiatomaceous earth using essentially the above-described titaniumdioxide deposition process, with the exception that a solution of 10.0 gof Fe(NO₃)₃.9H₂O (J. T. Baker, Inc., Phillipsburg, N.J.) and 25.0 g ofTiO(SO₄).2H₂O (Noah Technologies Corporation, San Antonio, Tex.)dissolved in 175 mL of deionized water was substituted for the titanylsulfate solution. A portion of the resulting iron oxide- and titaniumdioxide-modified diatomaceous earth was similarly calcined to 350° C.for further testing.

Microorganism Concentration Test Method

An isolated microorganism colony was inoculated into 5 mL BBL™Trypticase™ Soy Broth (Becton Dickinson, Sparks, Md.) and incubated at37° C. for 18-20 hours. This overnight culture at ˜10⁹ colony formingunits per mL was diluted in adsorption buffer (containing 5 mM KCl, 1 mMCaCl₂, 0.1 mM MgCl₂, and 1 mM K₂HPO₄) at pH 7.2 to obtain 10³microorganisms per mL dilution. A 1.1 mL volume of the microorganismdilution was added to separate, labeled sterile 5 mL polypropylene tubes(BD Falcon™, Becton Dickinson, Franklin Lakes, N.J.) containing 10 mg ofconcentration agent, each of which was capped and mixed on a ThermolyneMaximix Plus™ vortex mixer (Barnstead International, Iowa). Each cappedtube was incubated at room temperature (25° C.) for 15 minutes on aThermolyne Vari Mix™ shaker platform (Barnstead International, Iowa).After the incubation, each tube was allowed to stand on the lab benchfor 10 minutes to settle the concentration agent. Control sample tubescontaining 1.1 mL of the microorganism dilution without concentrationagent were treated in the same manner. The resulting settledconcentration agent and/or supernatant (and the control samples) werethen used for analysis.

The settled concentration agent was re-suspended in 1 mL sterileButterfield's Buffer solution (pH 7.2±0.2; monobasic potassium phosphatebuffer solution; VWR Catalog Number 83008-093, VWR, West Chester, Pa.)and plated on 3M™ Petrifilm™ Aerobic Count Plates culture medium (dry,rehydratable; 3M Company, St. Paul., MN) according to the manufacturer'sinstructions. Aerobic count was quantified using a 3M™ Petrifilm™ PlateReader (3M Company, St. Paul., MN). Results were calculated using thefollowing formula:Percent CFU/mL in Re-suspended Concentration Agent=(number of coloniesfrom plated re-suspended concentration agent)/(number of colonies fromplated untreated control sample)×100(where CFU=Colony Forming Unit, which is a unit of live or viablemicroorganisms). Results were then reported in terms of percent captureof microorganisms by the concentration agent using the formula below:Capture Efficiency or Percent Capture=Percent CFU/mL in Re-suspendedConcentration Agent

For comparison purposes, in at least some cases 1 mL of the supernatantwas removed and plated undiluted or diluted 1:10 in Butterfield's Buffersolution and plated onto 3M™ Petrifilm™ Aerobic Count Plates culturemedium. Aerobic count was quantified using a 3M™ Petrifilm™ Plate Reader(3M Company, St. Paul., MN). Results were calculated using the followingformula:Percent CFU/mL in Supernatant=(number of colonies from platedsupernatant)/(number of colonies from plated untreated controlsample)×100(where CFU=Colony Forming Unit, which is a unit of live or viablemicroorganisms). When the microorganism colonies and the concentrationagent were similar in color (providing little contrast for the platereader), results were based upon the supernatant and were then reportedin terms of percent capture of microorganisms by the concentration agentusing the formula below:Capture Efficiency or Percent Capture=100−Percent CFU/mL in Supernatant

Examples 1-12 and Comparative Examples 1 and 2

Using the above-described microorganism concentration test method, 10 mgof various different surface-treated diatomaceous earth orsurface-treated calcined diatomaceous earth concentration agents(prepared as described above) and 10 mg of untreated diatomaceous earth(hereinafter, DE) were tested separately for bacterial concentrationagainst target microorganisms, the gram-negative bacterium Salmonellaenterica subsp. enterica serovar Typhimurium (ATCC 35987) and thegram-positive bacterium Staphylococcus aureus (ATCC 6538). The resultsare shown in Table 1 below.

TABLE 1 Percent Capture ± Example Concentration Standard No.Microorganism Agent Deviation C-1 Staphylococcus DE  54 ± 13 1Staphylococcus TiO₂-DE 94 ± 4 2 Staphylococcus Fe₂O₃—TiO₂-DE 96 ± 1 3Staphylococcus calcined Fe₂O₃—TiO₂- 100 ± 0  DE 4 StaphylococcusPt-calcined DE 99 ± 0 5 Staphylococcus Au—Fe₂O₃-DE 99 ± 0 6Staphylococcus Au-calcined DE 99 ± 0 C-2 Salmonella DE 45 ± 1 7Salmonella TiO₂-DE 86 ± 3 8 Salmonella Fe₂O₃—TiO₂-DE 88 ± 1 9 Salmonellacalcined Fe₂O₃—TiO₂- 89 ± 5 DE 10  Salmonella Pt-calcined DE 72 ± 1 11 Salmonella Au—Fe₂O₃-DE 91 ± 6 12  Salmonella Au-calcined DE 100 ± 0  13 Salmonella Au-DE 89 ± 2

Examples 14-19 and Comparative Example 3

Using the above-described microorganism concentration test method, 10 mgof various different surface-treated diatomaceous earth orsurface-treated calcined diatomaceous earth concentration agents(prepared as described above) and 10 mg of untreated diatomaceous earth(hereinafter, DE) were tested separately for yeast concentration of thetarget microorganism, Saccharomyces cerevisiae (10² CFU/mL; ATCC201390). The resulting materials were plated on 3M™ Petrifilm™ Yeast andMold Count Plate culture medium (dry, rehydratable; 3M Company, St.Paul, Minn.) and incubated for 5 days according to the manufacturer'sinstructions. Isolated yeast colonies were counted manually, and percentcapture was calculated as described above. The results are shown inTable 2 below (standard deviation for all samples less than 10 percent).

TABLE 2 Example Concentration No. Microorganism Agent Percent CaptureC-3 Saccharomyces DE 42 14 Saccharomyces TiO₂-DE 99 15 SaccharomycesFe₂O₃—TiO₂-DE 93 16 Saccharomyces calcined Fe₂O₃—TiO₂- 100 DE 17Saccharomyces Pt-calcined DE 100 18 Saccharomyces Au—Fe₂O₃-DE 100 19Saccharomyces Au-calcined DE 99

Examples 20-22 and Comparative Examples 4-6

Food samples were purchased from a local grocery store (Cub Foods, St.Paul). Ham slices, lettuce, and apple juice samples (11 g) were weighedin sterile glass dishes and added to sterile Stomacher™ polyethylenefilter bags (Seward Corp, Norfolk, UK). The food samples were spikedwith bacterial cultures at a 10² CFU/mL concentration using an 18-20hour overnight culture (stock) of Salmonella enterica subsp. entericaserovar Typhimurium (ATCC 35987). This was followed by the addition of99 mL of Butterfield's Buffer solution to each spiked sample. Theresulting samples were blended for a 2-minute cycle in a Stomacher™ 400Circulator laboratory blender (Seward Corp. Norfolk, UK). The blendedsamples were collected in sterile 50 mL conical polypropylene centrifugetubes (BD Falcon™, Becton Dickinson, Franklin Lakes, N.J.) andcentrifuged (Eppendorf™ centrifuge 5804; Westbury, N.Y.) at 2000revolutions per minute (rpm) for 5 minutes to remove large debris. Theresulting supernatants were used as samples for further testing.

Using the above-described microorganism concentration test method, each1 mL test sample prepared as above was added separately to a test tubecontaining 10 mg of surface-treated diatomaceous earth and to a controltest tube containing 10 mg of untreated diatomaceous earth and testedfor bacterial concentration of the target microorganism, Salmonellaenterica subsp. enterica serovar Typhimurium (ATCC 35987). For testingin lettuce, samples were placed in sterile 100×20 mm tissue culturepetridishes (Sarstedt, Newton, N.C.) and incubated under ultraviolet(UV) lights in an AlphaImager™ MultiImage™ light cabinet (Alpha InnotechCorporation, San Leandro, Calif.) for 1 hour to eliminate backgroundflora. Such UV-treated samples were confirmed for absence of nativeflora (by plating and counting a 1 mL sample essentially as describedabove) and then used for concentration experiments. The results areshown in Table 3 below (standard deviation for all samples less than 10percent).

TABLE 3 Concentration Percent Example No. Microorganism Agent SampleCapture C-4 Salmonella DE Apple Juice 43 20 Salmonella Au—Fe₂O₃-DE AppleJuice 94 C-5 Salmonella DE Ham 75 21 Salmonella Au—Fe₂O₃-DE Ham 94 C-6Salmonella DE Lettuce 55 22 Salmonella Au—Fe₂O₃-DE Lettuce 80

Examples 23-24 and Comparative Examples 7-8

Following the procedure of Examples 20-22 and Comparative Examples 4-6above with a turkey sample (using 25 g of sliced turkey and 225 mLButterfield's Buffer solution), surface treated diatomaceous earth anduntreated diatomaceous earth were separately tested for concentration ofthe target microorganism Salmonella enterica subsp. enterica serovarTyphimurium (ATCC 35987) from large-volume samples (300 mg concentrationagent per 30 mL sample volume). Also tested was potable water (100 mL)from a drinking fountain, which was collected in a sterile 250 mL glassbottle (VWR, West Chester, Pa.) and inoculated with the targetmicroorganism Salmonella enterica subsp. enterica serovar Typhimurium(ATCC 35987) at 10² CFU/mL. The resulting inoculated water was mixedmanually end-to-end 5 times and incubated at room temperature (25° C.)for 15 minutes.

30 mL samples prepared as described above were added to sterile 50 mLconical polypropylene centrifuge tubes (BD Falcon™, Becton Dickinson,Franklin Lakes, N.J.) containing 300 mg of concentration agent and weretested by using the above-described microorganism concentration testmethod. The resulting settled concentration agent was re-suspended in 30mL sterile Butterfield's Buffer solution and plated on 3M™ Petrifilm™Aerobic Count Plates culture medium. The results are shown in Table 4below (standard deviation for all samples less than 10 percent).

TABLE 4 Concentration Percent Example No. Microorganism Agent SampleCapture C-7 Salmonella DE Potable 79 Water 23 Salmonella Au—Fe₂O₃-DEPotable 97 Water C-8 Salmonella DE Turkey 52 24 Salmonella Au—Fe₂O₃-DETurkey 88

Examples 25-32

10 mg samples of various different surface-treated diatomaceous earthconcentration agents (prepared as described above) were testedseparately for concentration of the target bacterial endospores Bacillusatrophaeus (ATCC 9372) and Bacillus subtilis (ATCC 19659). Theabove-described microorganism concentration test method was utilizedwith the following modifications: the overnight cultures had 1.4×10³CFU/mL Bacillus atrophaeus and 6×10² CFU/mL Bacillus subtilis,respectively; the resulting supernatants were plated undiluted; and thesettled concentration agent with bound microorganism was resuspended in5 mL sterile Butterfield's Buffer solution and plated in duplicate (1 mLeach). Capture efficiencies were calculated based on counts from theplated supernatants, and the results are shown in Table 5 below(standard deviation for all samples less than 10 percent).

TABLE 5 Concentration Example No. Microorganism Agent Percent Capture 25Bacillus atrophaeus TiO₂-DE 79 26 Bacillus atrophaeus Pt-DE 100 27Bacillus atrophaeus Au-DE 81 28 Bacillus atrophaeus Au—Fe₂O₃-DE 100 29Bacillus subtilis TiO₂-DE 97 30 Bacillus subtilis Pt-DE 100 31 Bacillussubtilis Au-DE 99 32 Bacillus subtilis Au—Fe₂O₃-DE 99

Examples 33-36

10 mg samples of two different surface-treated diatomaceous earthconcentration agents (namely, Pt-DE and Au—Fe₂O₃-DE) were testedseparately for concentration of the target non-enveloped,bacteria-infecting virus, Escherichia coli bacteriophage MS2 (ATCC15597-B1; which is often used as a surrogate for varioushuman-infecting, non-enveloped enteric viruses). A double layer agarmethod (described below) was used to assay for capture of theEscherichia coli bacteriophage MS2 (ATCC 15597-B1) using Escherichiacoli bacteria (ATCC 15597) as host.

Escherichia coli bacteriophage MS2 stock was diluted ten-fold seriallyin sterile 1× adsorption buffer (containing 5 mM KCl, 1 mM CaCl₂, 0.1 mMMgCl₂, and 1 mM K₂HPO₄) at pH 7.2 to obtain two dilutions with 10³ and10² plaque forming units per milliliter (PFUs/mL), respectively. A 1.0mL volume of resulting bacteriophage dilution was added to a labeledsterile 5 mL polypropylene tube (BD Falcon™, Becton Dickinson, FranklinLakes, N.J.) containing 10 mg of concentration agent and mixed on aThermolyne Maximix Plus™ vortex mixer (Barnstead International, Iowa).The capped tube was incubated at room temperature (25° C.) for 15minutes on a Thermolyne Vari Mix™ shaker platform (BarnsteadInternational, Iowa). After the incubation, the tube was allowed tostand on the lab bench for 10 minutes to settle the concentration agent.A control sample tube containing 1.0 mL of the bacteriophage dilutionwithout concentration agent was treated in the same manner. Theresulting settled concentration agent and supernatant (and the controlsample) were then used for analysis.

100 microliters of the supernatant was removed and assayed forbacteriophage using the double layer agar method described below. Anadditional 800 microliters of supernatant was removed and discarded. Onehundred microliters of the settled concentration agent was also assayedfor bacteriophage.

Double Layer Agar Method:

A single colony of Escherichia coli bacteria (ATCC 15597) was inoculatedinto 25 mL sterile 3 weight percent tryptic soy broth (Bacto™ TrypticSoy Broth, Becton Dickinson and Company, Sparks, Md.; prepared accordingto manufacturer's instructions) and incubated at 37° C. in a shakerincubator (Innova™ 44, New Brunswick Scientific Co., Inc., Edison, N.J.)set at 250 revolutions per minute (rpm) overnight. 750 microliters ofthis overnight culture was used to inoculate 75 mL sterile 3 weightpercent tryptic soy broth. The resulting culture was incubated at 37° C.in the shaker incubator set at 250 rpm to obtain Escherichia coli cellsin the exponential phase as measured by absorbance at 550 nm (absorbancevalues 0.3-0.6) using a SpectraMax M5 spectrophotometer (MolecularDevices, Sunnyvale, Calif.). The cells were incubated on ice until usedfor assay.

One hundred microliters of the above-described bacteriophage testsamples were mixed with 75 microliters of the ice-incubated Escherichiacoli (host bacteria) cells and incubated at room temperature (25° C.)for 5 minutes. The resulting samples were mixed with 5 mL sterile moltentop agar (3 weight percent tryptic soy broth, 1.5 weight percent NaCl,0.6 weight percent agar; prepared that day and maintained in a 48° C.waterbath). The mixture was then poured on top of bottom agar (3 weightpercent tryptic soy broth, 1.5 weight percent NaCl, 1.2 weight percentagar) in petridishes. The molten agar component of the mixture wasallowed to solidify for 5 minutes, and the petridishes or plates wereinverted and incubated at 37° C. The plates were visually inspectedafter overnight incubation, and those plates containing settledconcentration agent (as well as the control plate) showed the presenceof bacteriophage plaques. Capture efficiencies were calculated based oncounts from the plated supernatants and determined to be 96 percent and97 percent for Pt-DE (for the 10³ and 10² PFU/mL dilutions,respectively) and 94 percent and 95 percent for Au—Fe₂O₃-DE (for the 10³and 10² PFU/mL dilutions, respectively) (standard deviation less than 10percent).

Examples 37-38

Apple juice was purchased from a local grocery store (Cub Foods, St.Paul). Apple juice (11 g) was weighed in a sterile glass dish and addedto 99 mL sterile Butterfield's Buffer. The resulting combination wasmixed by swirling for 1 minute and was spiked with two bacterialcultures, each at a 1 CFU/mL concentration, using 18-20 hour overnightcultures (bacterial stocks) of Salmonella enterica subsp. entericaserovar Typhimurium (ATCC 35987) and Escherichia coli (ATCC 51813).Serial dilutions of the bacterial stocks had been made in 1× adsorptionbuffer as described above.

Using the above-described microorganism concentration test method, a 10mL volume of the spiked apple juice sample was added to a sterile 50 mLconical polypropylene centrifuge tube (BD Falcon™, Becton Dickinson,Franklin Lakes, N.J.) containing 100 mg of a surface-treateddiatomaceous earth concentration agent (namely, TiO₂-DE or Au—Fe₂O₃-DE)and incubated for 15 minutes for bacterial capture/concentration of thetarget microorganism, Salmonella (in the presence of the Escherichiacoli, a competitor microorganism). The resulting supernatant wasremoved, and the settled concentration agent was transferred to anothersterile 50 mL tube containing 2 mL sterile 3 weight percent tryptic soybroth (Bacto™ Tryptic Soy Broth, Becton Dickinson and Company, Sparks,Md.; prepared according to manufacturer's instructions). The tube wasloosely capped, and its contents were mixed and incubated at 37° C.After overnight incubation, the resulting broth mixture was tested forthe presence of Salmonella using a RapidChek™ Salmonella lateral flowimmunoassay test strip from SDI (Strategic Diagnostics, Inc., Newark,Del.). Visual inspection of the test strip showed it to be positive forSalmonella.

Nucleic acid detection by polymerase chain reaction (PCR) was alsocarried out for the microorganism-containing broth mixture. 1 mL of theabove-described overnight-incubated, concentration agent-containingbroth was assayed as a test sample for the presence of Salmonella byusing a TaqMan™ ABI Salmonella enterica Detection Kit from AppliedBiosystems (Foster City, Calif.). As a control sample, 1 mL of the 18-20hour overnight culture (stock) of Salmonella enterica subsp. entericaserovar Typhimurium (ATCC 35987) was also assayed. PCR testing wasconducted in a Stratagene Mx3005P™ QPCR (quantitative PCR) System(Stratagene Corporation, La Jolla, Calif.) by using the following cycleconditions per cycle for 45 cycles: 25° C. for 30 seconds, 95° C. for 10minutes, 95° C. for 15 seconds, and 60° C. for 1 minute. An average(n=2) cycle threshold value (CT value) of 17.71 was obtained for thecontrol sample. Average (n=2) CT values of 20.44 and 16.53 were obtainedfor the test samples containing TiO₂-DE or Au—Fe₂O₃-DE, respectively,indicating a positive PCR reaction and confirming the presence ofSalmonella.

The referenced descriptions contained in the patents, patent documents,and publications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousunforeseeable modifications and alterations to this invention willbecome apparent to those skilled in the art without departing from thescope and spirit of this invention. It should be understood that thisinvention is not intended to be unduly limited by the illustrativeembodiments and examples set forth herein and that such examples andembodiments are presented by way of example only, with the scope of theinvention intended to be limited only by the claims set forth herein asfollows:

We claim:
 1. A process comprising (a) providing a concentration agentthat comprises diatomaceous earth bearing, on at least a portion of itssurface, a surface treatment comprising a surface modifier comprisingtitanium dioxide, fine-nanoscale gold or platinum, or a combinationthereof; (b) providing a sample comprising at least one microorganismstrain; (c) contacting said concentration agent with said sample suchthat said at least one microorganism strain is bound to or captured bysaid concentration agent, such that the at least one microorganismstrain remains viable; and (d) detecting the presence of at least onebound microorganism strain.
 2. The process of claim 1, wherein saidconcentration agent is a particulate concentration agent.
 3. The processof claim 1, wherein said surface modifier is selected fromfine-nanoscale gold, fine-nanoscale platinum, fine-nanoscale gold incombination with at least one metal oxide, titanium dioxide, titaniumdioxide in combination with at least one other metal oxide, andcombinations thereof.
 4. The process of claim 3, wherein said metaloxide is selected from ferric oxide, titanium dioxide, zinc oxide,aluminum oxide, and combinations thereof.
 5. The process of claim 3,wherein said surface modifier is selected from fine-nanoscale gold,fine-nanoscale platinum, fine-nanoscale gold in combination with atleast ferric oxide or titanium dioxide, titanium dioxide, titaniumdioxide in combination with at least ferric oxide, and combinationsthereof.
 6. The process of claim 1, wherein said sample is in the formof a fluid.
 7. The process of claim 1, wherein said microorganism strainis selected from strains of bacteria, fungi, yeasts, protozoans,viruses, bacterial endospores, and combinations thereof.
 8. The processof claim 7, wherein said microorganism strain is selected from strainsof bacteria, yeasts, viruses, bacterial endospores, and combinationsthereof.
 9. The process of claim 1, wherein said contacting is carriedout by mixing said concentration agent and said sample.
 10. The processof claim 1, wherein said detecting is carried out by a method selectedfrom culture-based methods, microscopy and other imaging methods,genetic detection methods, immunologic detection methods,bioluminescence-based detection methods, and combinations thereof. 11.The process of claim 1, wherein said process further comprisessegregating the resulting microorganism-bound concentration agent. 12.The process of claim 11, wherein said segregating is effected by amethod selected from gravitational settling, centrifugation, filtration,and combinations thereof.
 13. The process of claim 12, wherein saidsegregating is effected at least in part by gravitational settling. 14.The process of claim 11, wherein said process further comprisesseparating the resulting segregated concentration agent from saidsample.
 15. A kit comprising (a) a concentration agent that comprisesdiatomaceous earth bearing, on at least a portion of its surface, asurface treatment comprising a surface modifier comprising titaniumdioxide, fine-nanoscale gold or platinum, or a combination thereof; and(b) a testing container for use in carrying out the process of claim 1.